This R21 Developmental Research Grant harnesses multi-photon laser technology to image a working neuronal network in vitro, and then test how constituent neurons contribute to network function via cell-specific laser ablation. Specifically, a computer-controlled system has been developed to detect neurons using multi-photon fluorescence microscopy, store the locations of these cells in memory, and then laser-ablate the target neurons one at a time, in sequence, while monitoring the function of the neural circuit electrophysiologically. Use of a long- wavelength pulsed laser provides unprecedented specificity and control of the lesion in three- dimensional tissue. This significant technological development provides a powerful new tool for interrogating the cellular bases for network behaviors, as well as pathophysiological breakdown in network function. This project focuses on the neural generation and control of breathing behavior. In particular, the investigations probe the circuit properties of the specialized inspiratory rhythm-generating site called the preBotzinger Complex (preBotC) located in the lower brainstem of humans and all mammals. SPECIFIC AIM 1 will evaluate the cellular composition of the preBotC. Transgenic mouse models will be used to apply fluorescent tags to genetically distinct subpopulations of neurons in the preBotC, and then selectively and serially lesion them to test their respective roles in rhythmogenesis. The hypothesis that neurons derived from the homeodomain transcription factor Dbx1 comprise the essential rhythm- generating kernel of the preBotC will be specifically tested. SPECIFIC AIM 2 will evaluate whether and how respiratory function deteriorates when rhythmically active preBotC neurons are sequentially deleted. Here the target neurons will be selected on the basis of inspiratory rhythmic activity, rather than genetic origin. This set of experiments will serve as a general disease model to examine the cellular mechanisms underlying respiratory pathologies that have a central etiology. In summary, this R21 project will provide significant new information regarding the neural generation and control of breathing. This new knowledge is important for human health and wellness given that breathing is a relentless and indispensable human behavior that maintains homeostasis and life itself. In subsequent projects this new technique - for detecting and then ablating neurons in a cumulative sequence in vitro - will be applied to interrogate locomotor and masticatory rhythm-generating networks that can be also be studied in spinal cord and hindbrain preparations in vitro. Indeed, this lesioning system will be broadly applicable to studying networks in vitro from any brain region. PUBLIC HEALTH RELEVANCE: Breathing is a vital human behavior that is essential to maintain homeostasis and life itself. This project will advance our understanding of the cellular composition of brainstem neural circuits that generate and control breathing rhythms, and examine how respiratory function breaks down as respiratory rhythm-generating neurons progressively die. The new knowledge obtained will serve as a foundation for the treatment and prevention of respiratory disorders with a central neural etiology, and elucidate circuit-level properties that underlie rhythmic motor behaviors in general.